Human prostatic cell lines immortalized by adenovirus 12-simian virus 40 (AD12/SV40) hybrid virus

ABSTRACT

Immortalized non-tumorigenic or tumorigenic human prostatic epithelial and fibroblast cell lines and derivatives thereof, containing DNA of a hybrid virus between adenovirus 12 and simian virus 40. The cell lines are useful for research on causes, treatment and prevention of prostate cancer, benign prostatic hyperplasia, male infertility, birth defects, aging and assessment of environmental toxic agents.

This is a divisional of application Ser. No. 08/234,981 filed on Apr.28, 1994 now U.S. Pat. No. 5,610,043.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a novel immortalized epithelial cellline containing a hybrid virus and method for its preparation. Inparticular the present invention relates to a hybrid virus derived fromadenovirus 12 and simian virus 40 viruses which is then used for theimmortalization of the epithelial cells.

(2) Description of Related Art

Human cells are generally difficult to grow and maintain in long-termcultures in vitro. They have a limited life span in culture, grow for ashort time and usually after 4 or 5 subcultures, they senescence anddie.

Prostate cancer is the leading cancer in men in the United States, interms of incidence. Thirty-two percent (32%) of all cancers in men arisein the prostate. It is estimated that 200,000 new cases of prostatecancer will occur in the U.S. in 1994. Prostate cancer is the secondleading cause of death from cancer and 38,000 deaths are estimated tooccur in 1994 (Boring et al., CA, Cancer J. Clin. 44:7, 1994). AfricanAmerican men have the highest incidence of prostate cancer in the world,which is almost twice as high as that in white men and more than 600times higher than in men from Thailand (Webber et al., In Vitro Modelsfor Cancer Res. Vol. V, pp. 3-24, Boca Raton, CRC Press, 1988). One in10 men in the U.S. will develop prostate cancer in their lifetime (byage 85). An estimated 11 million men have latent or clinical prostaticcarcinoma. Sixty-five percent (65%) of the cases already have metastaticdisease at the time of diagnosis. The survival rate is less than 20%.

The causes of prostate cancer are not known at the present time. A studyof the causes, prevention and treatment has been hampered by the factthat good animal or cell models are not available. Although ratprostatic cells have been used extensively for such studies, ratprostate is not homologous to the human prostate, thus, it is not anideal system to use.

There is a need for cell lines derived from normal human prostate whichcan be used for studies on the process of prostate cancer development inman and to identify agents which may cause or prevent prostate cancer.

Attempts have been made to immortalize human adult prostatic epithelialcells using a monkey virus (Simian virus SV40; Cussenot, O., et al.,Journal of Urology 143, 881-886 (1991); Kaighn, M. E., et al, CancerResearch, 49, 3050-3056 (1989); Lee et al., Internat. J. Oncol. 4, 821(1994)).

Infection of mammalian cells with Simian virus 40 (SV40) can have twooutcomes. In some simian cells which allow multiplication of the virusand are hence called "permissive cells", infection results in virusproduction and ultimately in cell death. Cells which do not permit virusmultiplication are designated as "non-permissive" cells, such as rodentcells. Infection of these cells leads to induction and maintenance of analtered "immortalized" phenotype at a low frequency. Invariably, anintegrated copy of vital DNA (vDNA) persists.

The viral DNA in non-permissive, SV40-immortalized cells usually cannotbe induced to excise itself and replicate, but virus can be induced toreplicate after fusion of non-permissive cells with permissive simiancells. According to Gish et al (Gish, W. R., et al., J. Virol.,61:2864-2876 (1987)), two essential viral components for this excisionand lytic replication are cis-acting origin of replication (ori) and areplication component A gene product (large T antigen) acting in trans.

In culture, many types of human cells are semi-permissive for SV40replication. The semi-permissive response of human cells to SV40infection appears to be complex. Human cells can be productivelyinfected by the virus, but they yield 100 times less virus than thesimian cells. A few cells, which survive the lytic infection, may passthrough a crisis period to yield immortalized cell lines which carryintegrated copies of vDNA (Gish, W. R., et al., J. Virol., 61:2864-2876(1987)). During this period, the rate of cell multiplication is markedlyreduced or ceases. It has also been observed that cultures ofSV40-infected human cells that were nursed through crisis no longerproduced infectious SV40, although they had done so prior to crisis(Girardi, A. J., et al., J. Cell. Comp. Physiol. 65:69-84 (1965)).Furthermore, survivors of crisis exhibited higher levels of T-antigenstaining than they did prior to crisis. High levels of T-antigen may benecessary for survival of cells through crisis (Girardi, A. J., et al.,J. Cell. Comp. Physiol. 65:69-84 (1965)).

Three human cell lines immortalized only by SV40 virus alone have beendeveloped in recent years. Immortalization of human neonatal prostateepithelial cells by strontium phosphate transection method using aplasmid (pRSV-T) containing SV40 was first accomplished by Kaighn etal., (Kaighn, M. E., et al., Cancer Res., 49:3050-3056 (1989)). Theplasmid pRSV-T was developed at the National Cancer Institute. It is anSV40 ori construct containing the SV40 early region genes and the Rousesarcoma virus long terminal repeat (Gorman, C., et al., Science,221:551-553 (1983)). These cells formed rapidly growing, multi-layeredcolonies within two weeks and according to the authors, there was littleor no indication of crisis. These cells contain cytokeratins and SV40-Tantigen. They do not produce tumors in nude mice.

Cussenot et al (Cussenot, O., et al., J. Urol., 143:881-886 (1991)) useda plasmid containing SV40 genome with defective replication origin(ori-) encapsulated into liposomes. The cells were shown to contain theSV40 genome. They express large T-antigen and are positive forcytokeratins (CK) 18 and 19, weekly positive for prostatic acidphosphatase (PAP) and prostate specific antigen (PSA) and negative forCK 14. These cells contain high affinity receptors for5α-dihydrotestosterone (5α-DHT).

In work similar to that of Cussenot et al., (J. Urol. 143:881-886,1991), Lee et al., (Internat. J. Oncol. 4:821, 1994) used a plasmid(pRNS-1) containing an origin-defective SV40 genome and a plasmidcarrying the neomycin resistance gene. A cell line was established,however, this cell line has not been characterized with regard to itsprostatic epithelial origin on the basis of androgen responsiveness.These investigators were unsuccessful in establishing an immortalizedprostatic epithelial cell line using Ad12-SV40 hybrid virus.

Adenoviruses have the ability to extend the lifespan of mammalian cells.When rat embryo cells are infected with adenovirus-2 (AD-2) virus, afraction of the cells show extended lifespan but infectious virus couldnot be isolated from them although vital DNA persisted, as recognized byhybridization with labelled probes (Gallimore, P. H., et al., J. Mol.Biol. 89:49-72 (1974)).

In 1964, Rabson et al., (Proc. Soc. Exper. Biol. Med. 116:187-190,1964), reported that the growth of adenoviruses in African green monkeykidney (AGMK) cell cultures is enhanced following pre-infection withSV40 virus. Schell et al (Schell, K., et al., Proc. Natl. Acad. Sci.USA, 55:81-88 (1966)) observed that the oncogenicity was markedlyenhanced when adenovirus-12 (AD-12) and SV40 were passed seriallytogether for five or more passages in AGMK cells. During the course oftheir investigation, Schell et al., (Schell, K., et al., Proc. Natl.Acad. Sci. USA, 55:81-88 (1966)) found that hybridization of the twoviruses occurred. This hybrid virus was used in the present invention.This is the first successful immortalization of human prostaticepithelial cells with Ad12-SV40 hybrid virus.

OBJECTS

It is therefore an object of the present invention to provide animmortalized normal human prostatic epithelial cell line. Further, it isan object of the present invention to provide an epithelial cell linewhich is useful for research in vitro. A further objective of thepresent invention is a method for conversion of a non-tumorigenic,immortalized cell into a tumorigenic cell by the introduction of anoncogene. Further, the present invention relates to methods and kits forscreening carcinogenic agents or potential chemotherapeutic,chemopreventive, anti-invasive and anti-metastatic agents using animmortalized adult human prostate epithelial cell line. These and otherobjects will become increasingly apparent by reference to the followingdescription and the drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of a secondary culture of prostatic epitheliumthree months after infection with Ad12-SV40 virus. It shows two clones.PWR-1E cell line is a subclone of one of these.

FIG. 1B is an uninfected control culture in which all cells havesenescenced.

FIG. 2 is a photograph showing subcloning of a clone of epithelial cellsdescribed in FIG. 1A. This was done to ensure selection of a true clone.A sample plate (shown here) was fixed and stained with crystal violet 15days after plating and cloning efficiency was also determined.

FIG. 3 is a microscopic photograph of PWR-1E cells in passage 30,stained with hematoxylin and eosin. These cells have an epithelialmorphology (625X).

FIG. 4 is a graph showing growth of PWR-1E cells. Cells were plated atdensities varying from 625 cells/well to 40,000 cells/well, in 96 wellplates. Plates were fixed and stained with Methylene blue and absorbancemeasured at 620 nm earlier (Cai et al., Exper. Cell Res. 192:366, 1991).

FIG. 5 is a graph showing the growth of PWR-1E in the presence of5α-dihydrotestosterone (5α-DHT). Ten thousand (10,000) cells were platedper well of a 96-well microplate and exposed to varying concentration of5α-DHT (10⁻¹² M to 10⁻⁵ M). 5α-DHT was dissolved in ethanol. PWR-1Ecells show a growth response to 5α-DHT in culture.

FIGS. 6A to 6D consist of microscopic photographs showingimmunolabelling of cellular proteins in PWR-1E cells. Proteins weredetected by immuno-avidin-biotin-peroxidase staining.

FIG. 6A is a microscopic photograph showing strong positive staining forcytokeratin 8.

FIG. 6B is a microscopic photograph showing strong positive staining forcytokeratin 18. This staining demonstrates that PWR-1E cells are ofepithelial origin showing expression of cytokeratins characteristic ofluminal prostatic epithelial cells.

FIG. 6C is a microscopic photograph showing absence of staining withantibody to desmin. Desmin is a cytoskeletal protein expressed in musclecells. This absence of staining further demonstrates that PWR-1E cellsare epithelial and not muscle cells.

FIG. 6D is a microscopic photograph showing absence of staining withantibody to Factor VIII. Factor VIII is expressed by endothelial cells.This absence of staining further demonstrates that PWR-1E cells areepithelial and not endothelial cells (625X).

Southern blot analysis shows that PWR-1E carry the SV40 large T-antigengene.

PWR-1E cells are not able to form colonies in soft agar. Thisdemonstrates that, like normal cells, PWR-1E are anchorage dependent.

Ten (10) million PWR-1E cells were injected subcutaneously into nudemice. After 4 months none of the animals (0/3) developed tumors. Thisdemonstrates that PWR-1E cells are not tumorigenic.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to an immortalized human prostaticepithelial or fibroblast cell line in culture, free of other cell lines,containing DNA of adenovirus and simian virus as a hybrid virus. Thisfurther includes immortalization and transformation with adenovirus E1Aand E1B and simian virus 40 middle-T and large-T antigen genes. Thecells can be made tumorigenic by introducing an activated, viral Ki-rasoncogene in the cells.

In particular, the present invention relates to a method for producingimmortalized human prostatic epithelial or fibroblast cell lines whichcomprises: providing human prostatic epithelial or fibroblast cells; andinfecting the cells with a hybrid virus derived from an adenovirus and asimian virus to thereby immortalize the cells.

Further, the present invention relates to kit for screeningcarcinogenic, chemotherapeutic or chemopreventive agents comprising animmortalized human prostate epithelial cell line or derivative thereofcontaining DNA of adenovirus and simian virus as a hybrid virus.

Finally, the present invention relates to a method for testingcarcinogenicity of an agent comprising culturing the hybridvirus-containing cell line with an agent suspected of being carcinogenicand determining formation of an abnormal cellular mass by said cellline, the formation of the abnormal cellular mass being indicative ofcarcinogenicity of said agent.

The epithelial cells containing the hybrid virus (PWR-1E) are depositedon 7 Apr. 1994 under the Budapest Treaty with the American Type CultureCollection in Rockville, Md. as ATCC CRL 11611. The adenovirus 12 is apublicly available deposit ATCC VR-863, is also described in Schell etal., Proc. Nat. Acad. Sci. 55:81-88, 1966, and is available from manyresearch sources. The simian virus 40 is a publicly available depositATCC VR-239, is also described in Schell et al., Proc Nat. Acad. Sci.55:81-88, 1966 and is available from many research sources.

The PWR-1E cells are of prostatic epithelial origin and expresscytokeratins 8 and 18, which demonstrates their origin from thedifferentiated secretory cells. They do not express desmin, whichfurther demonstrates their epithelial origin and excludes origin frommuscle cells. They do not express Factor VIII, which excludes originfrom endothelial cells. They were negative for virus production in a"cytopathic effect assay" in Vero cells (Rhim et al., Science227:1250-1252, 1985). They show a moderate growth response to 5α-DHT andearly passages, without androgen stimulation, were weakly positive forprostate specific antigen (PSA). Preliminary results show that thesecells have a normal profile for the expression of plasminogen activators(urokinase and tissue-type plasminogen activator) and type IVcollagenases.

EXAMPLE 1

1. Isolation and culture of prostatic cells

Methods for isolation and maintenance of pure cultures of humanprostatic epithelium were developed by Webber, (Webber, M. M., In vitromodels for prostatic cancer: Summary. In: Models for prostate cancer, G.P. Murphy (ed.) pp. 133-147, New York, N.Y.: Alan R. Liss. (1980);Webber, M. M., Normal and benign human prostatic epithelium in culture.In Vitro, 15: 967-982 (1979); Webber, M. M., In Vitro Models for CancerResearch, Vol. V, pp. 25-125, Boca Raton, Fla.: CRC Press (1988);Chapronier-Rickenberg, D. M. et al., Growth of Cells in HormonallyDefined Media, 9B:1109-1115, (1982)).

a. Isolation

Isolation and growth of human prostatic epithelium was accomplishedaccording to methods described previously. The medium is serum-free andchemically defined. These studies have been described in detailelsewhere (Webber, M. M., In vitro models for prostatic cancer: Summary.In: Models for prostate cancer, G. P. Murphy (ed.) pp. 133-147, NewYork, N.Y.: Alan R. Liss. (1980); Webber, M. M., Normal and benign humanprostatic epithelium in culture. In Vitro, 15:967-982 (1979); Webber, M.M., In Vitro Models for Cancer Research (Vol. V), pp. 25-125, BocaRaton, Fla.: CRC Press (1988); and Chaproniere-Rickenberg, D. M., etal., A chemically defined medium for the growth of adult human prostaticepithelium. Cold Spring Harbor Symp. on "Growth of Cells in HormonallyDefined Media.", 9B:1109-1115, (1982)).

The normal primary cultures and earlier passages of PWR-1E cells weregrown in the serum-free KGM medium from Clonetics, San Diego, Calif.,supplemented with antibiotic/antimycotic mixture from GIBCO 600-5240 PG,Grand Island, N.Y. (Penicillin 100U, Streptomycin 100 μg and Fungizone25 μg,). Recent passages have been maintained in K-SFM, a serum-free,defined medium and antibiotic/antimycotic mixture from GIBCO K-SFM, No.10005-018, Grand Island, N.Y. Cells have been frozen successfully andrecovered with good viability. A tissue specimen of normal prostate froma 67 year old white male patient undergoing surgery for bladder cancerwas used for epithelial cell isolation. Pathology report stated "normalprostate, mild nodular hyperplasia, no neoplasms". The tissue was cutinto 2-3 mm cubes and placed in one 100 mm Petri dish with 30 mlRPMI-1640 containing 5% FBS (Intergen, No. 1050-75, Purchase, N.Y.), and400 Units/ml of collagenase (GIBCO Cat. No. 840-701811, Grand Island,N.Y.) activity 196 U/mg, antibiotics/ml as follows: Penicillin 100U,Streptomycin 100 μg and Fungizone 25 μg, Gentamycin 10 μg). After 48 hr.digestion in collagenase, the tissue was triturated until all lumps werebroken up, and centrifuged to remove collagenase. The pellet wassuspended in Saline G and allowed to settle in the refrigerator for 1hour. The sediment was suspended again and allowed to settle at 4° C.Four such washes were done. The sediment, after the last wash,containing the acini was suspended in storage medium containing spermine(2 μg/ml) and left at 4° C. overnight to destroy any fibroblasts thatmay be present in the preparation (Webber, M. M., Cell Biol. Internat.Rep., 4:185-194 (1980)).

b. Culture

The following day, the acini were centrifuged, suspended in growthmedium and plated. Primary cultures of prostatic epithelium wereestablished. Growth medium consisted of KGM (keratinocyte growth medium)from Clonetics No. CC-3001, San Diego, Calif. KGM has 0.2 mM calcium,0.1 ng/ml EGF and pituitary extract. This medium was supplemented with10 ng EGF/ml to make sure that the cells were not lost. Previous workhas shown that prostate epithelial cells grow well with this EGFsupplementation. To further ensure the survival of these cells, cultureflasks/plates for growing these cells were coated with a mixture of 10μg/ml each of fibronectin and collagen IV (Collaborative Research, No.40008 and No. 40233, respectively). During the past year, we have beenusing K-SFM medium from GIBCO, Grand Island, N.Y. for maintaining thecell lines. This medium contains 0.09 mM calcium, 50 μg/ml pituitaryextract and 5 ng/ml EGF with no other supplementation.

Primary cultures were subcultured using GIBCO Trypsin: EDTA mixture(0.05% trypsin, 0.02 mM EDTA, No. 25300-013) diluted 1:1 with PBS afterone wash with ca/Mg-free Dulbecco's PBS. Cells were plated in coatedplates. Medium was changed every third day. These cells were furthersubcultured to determine the number of passages until senescence andcell loss. These cells could not be propagated beyond five passages.

2. Immortalization by infection with AD12-SV40 hybrid virus

Primary and secondary cultures were used for this infection. AD12-SV40preparation (1:10 dilution) was used (Rhim, J. S., et al., Proc. Natl.Acad. Sci. USA, 78:313-317 (1981)). Several cultures were infected.Untreated control cultures were also maintained and medium was changedtwice weekly. After 10 days the infected cultures were subcultured andplated in coated plates. Cells were fed every third day. One month afterinfection, colonies began to appear and some areas of the culture showedcytopathic effects, whereas, the control cultures showed signs ofdegeneration. Two weeks later, clones from infected cultures wereisolated. These were plated into coated 24-well plates. Twenty-four (24)clones were isolated. One clone grew rapidly and was further propagatedthrough several passages. This clone did not show any cytopathic effectsand cells had an epithelial morphology. Cells from passage 10 wererecloned by plating 1000 cells per 100 mm plate. Cloning efficiency ofthis population was 4.4%. These clones were further subcloned bydilution plating of single cells in 96-well plates. Sixty-six (66)clones were isolated and further propagated.

FIG. 2 shows the second cloning of cells. From this cloning, anAD12-SV40 Immortalized, non-malignant cell line, designated as PWR-1Ecells has been established. At present, passage 35 cultures of PWR-1Ecells show epithelial morphology, and they are of a uniform size andshape. The cell sheets look excellent in terms of morphology. Thecultures do not show any cytopathic effects. These cells have undergoneover 250 cell generations.

The AD12-SV40 virus consists of recombinant DNA containing all or partof the SV40 genome and part or all of the adenovirus genome enclosed inadenovirus capsids. The AD12-SV40 hybrid virus was found to be highlyoncogenic in newborn hamsters.

An advantage of using AD12-V40 hybrid virus for immortalization is thatcultures infected with the hybrid virus escape the extended "crisis"period, lasting as long as several months, experienced by culturesinfected only with SV40 virus. This was demonstrated by Stoner et al.,(Stoner, G. D., et al., Cancer Res., 51:353-371 (1991)) using humanbronchial epithelial cells. However, these cells did not express theadenovirus early region proteins E1A and E1B. Cells immortalized withadenoviruses retain many of the characteristics of parent normal cells(Emami, S., et al., Proc. Natl. Acad. Sci. USA, 86:3194-3198 (1989)).

Immortalization with Ad12-SV40 hybrid virus is especially important whenone analyzes the process of immortalization and malignant transformationby oncogenic DNA viruses. Adenovirus and SV40 viruses encode certainproteins which are associated with the ability to immortalize and inducemalignant transformation.

Once the cells were immortalized, an extensive characterization ofimmortalized cells was essential before they could be used as a cellmodel system as follows.

EXAMPLE 2

1. Characterization of cells on the basis of intermediate filament andother proteins

PWR-1E cells were characterized first to establish their epithelialorigin on the basis of keratin expression. Expression of cytokeratins8/18 pair associated with prostatic epithelial cells was examined.PWR-1E cells showed strong expression of both cytokeratins 8 and 18. Thepresence of keratins 8 and 18 proves, beyond doubt, the epithelialorigin of PWR-1E cells and eliminates the possibility of fibroblasticorigin since fibroblasts do not express keratins.

Intermediate filaments (IFs) are an essential component of thedevelopmental program of different cell lineages (Sommers, C. L., etal., Cancer Res., 52:5190-5197 (1992)). By knowing the IF proteinsexpressed by cell lines, it becomes possible to characterize the originof the cells. For example, cells of simple epithelia coordinatelyexpress certain cytokeratins. Thus, IFs can serve as useful markers forcharacterization of cells. IFs are constituents of virtually alldifferentiated cells and are one of the components of the cellcytoskeleton expressed in a tissue specific manner (Sommers, C. L., etal., Cancer Res., 52:5190-5197 (1992)). Cells of epithelial originnormally express cytokeratins while those of mesenchymal origin expressvimentin IFs (Sommers, C. L., et al., Cancer Res., 52:5190-5197 (1992)).In addition to cytokeratin expression in PWR-1E cells, expression ofother IF proteins was also examined to make certain that our cell linedid not originate from any cell type other than prostate epithelium.

a. Cytokeratins

An important index of differentiation is the pattern of cytokeratinexpression. Keratins are expressed in a cell and tissue-specific mannerand their expression is known to change in abnormal differentiationprocesses. A human keratinocyte cell line KER-1 immortalized withlarge-T antigen gene shows a cytokeratin profile similar to that ofnon-immortalized keratinocytes except that KER-1 cells express higherlevels of cytokeratins 13 and 19 (Agarwal, C, et al., Cancer Res.,50:5947-5953 (1990)).

Normal epithelial cell differentiation is marked by the production ofdistinct cytokeratin IFs. They consist of a family of about 20polypeptides in the Mr range of 40 kDa to 70 kDa that are translatedfrom distinct mRNAs derived from a large multigene family. Allepithelial cells co-express at least one pair of cytokeratins,consisting of an acidic and a basic protein and these associate to formthe IFs (Molloy, C. J., et al., Exp. Mol. Pathol., 49:128-140 (1988)).As epithelial cells differentiate, additional keratins are expressed orsome may cease to be expressed. Thus, the appearance of specificcytokeratins is also a useful marker for identifying the level ofdifferentiation (O'Guin, W. M., Schermer, A., Lynch, M. and Sun, T.:Differentiation-specific expression of keratin pairs. In: Cellular andMolecular Biology of Intermediate Filaments, R. D. Goldman and P. M.Steinert eds.), New York, Plenum Press, pp. 301-334, 1990. Cytokeratinexpression can serve as a unique marker for prostatic epithelial celldifferentiation (Sherwood, E. R., et al., Prostate, 18: 303-314 (1991)).The prostatic epithelium is a simple epithelium, consisting of lessdifferentiated basal cells and more differentiated luminal cells with aspecialized secretory function (Sherwood, E. R., et al., Prostate, 18:303-314 (1991)). CK 8 and 18 have been identified as luminal cellspecific markers and CK 5 and 15 as basal cell specific marker pair. CK8, a 52 kDa basic protein, pairs with the acidic CK 18 (Mr 45 kDa).Overwhelming data show that the selective co-expression of CKs is highlyconserved in various epithelial lineages.

b. Desmin

To prove that the PWR-1E cells are not of smooth muscle origin,expression of desmin was examined. Epithelial cells should not expressdesmin. Desmin (Mr 53 kDa) IFs are found in smooth muscle cells (SMC),and in skeletal and cardiac muscle. A broad range of soft tissue tumors,e.g., satcomas express desmin but carcinomas and melanomas do not.Desmin has not been found in normal epithelia or epithelial tumors(Truong, L. D., et al., Am. J. Clin. Pathol., 93: 305-314 (1990)).PWR-1E cells were negative for desmin. This demonstrates that PWR-1Ecells are epithelial in origin.

2. Characterization on the basis of differentiated functions ofprostatic epithelial cells

a. Hormonal response

In vivo the growth and functional response of prostatic epithelium toandrogens is reflected by limited stimulation in cell proliferation andthe expression of PSA, PAP (Lin, M.-F., et al., Cancer Res.,52:4600-4607 (1992); and Fong, C.-J., et al., Prostate, 21:121-131(1992)). Secretion of both PSA and PAP responds to androgen stimulation.The presence of androgen receptor would be another marker forestablishing prostatic epithelial origin. Therefore, effect of 5α-DHT onthe growth of PWR-1E cells, the expression of PSA and PAP and thepresence of androgen receptor are being examined. It should however beemphasized that many transformed cell lines derived from hormoneresponsive normal cells lose hormone receptor and their ability torespond to that specific hormone. Examples include DU-145 and PC-3 humanprostate carcinoma cell lines (Webber, M. M., In vitro models forprostatic cancer: Summary. In: Models for prostate cancer, G. P. Murphy(ed.) pp. 133-147, New York, N.Y.: Alan R. Liss. (1980)).

Very few studies have been done on the effects of 5α-DHT on the growthof normal human prostatic epithelium. Prostatic epithelial cells willgrow in culture in the absence of androgen, however, they are androgenresponsive. In one of her earlier studies, Webber has shown (Webber, M.M., In vitro models for prostatic cancer: Summary. In: Models forprostate cancer, G. P. Murphy (ed.) pp. 133-147, New York, N.Y.: Alan R.Liss. (1980)) that the growth of normal human prostatic epithelium wasstimulated three to four fold in the presence of 0.3 μM 5α-DHT (Webber,M. M., Growth and maintenance of normal prostatic epithelium in vitro-Ahuman cell model. In: G. P. Murphy, A. A. Sandberg and J. P. Karr(eds.), Models for Prostate Cancer, pp. 181-216, New York: Alan R. Liss.(1980)). The majority of studies on the effects of androgens onprostatic cells in culture have been done using the prostatic carcinomacell line LNCaP. Generally in culture, 5α-DHT doses considerably higherthan those that induce cell proliferation cause increased secretion ofPSA and PAP proteins. For example, the earliest studies by Horoszewiczet al on LNCaP cells showed a 1.9 fold stimulation of cell growth with 1nM to 0.1 μM 5α-DHT in cultures containing charcoal stripped serum.These cells also contained 210 fmol androgen receptor/mg cytosol protein(Horoszewicz, J. S., et al., Cancer Res., 43: 1809-1818 (1983)). Theyobserved increase in PAP secretion at the entire range of 5α-DHT tested.However, much variation exists amongst results reported in differentstudies. Hasenson et al (Hasenson, M., et al., Prostate, 7:183-194(1985)) observed only a weak cell proliferation response in LNCaP cellsto 10 to 100 nM 5α-DHT and found only 16 fmol androgen receptor/mgprotein. de Launoit et al., (de Launoit, Y., et al., Cancer Res.,51:5165-5170 (1991)) observed a biphasic effect on LNCaP cellproliferation with a maximum stimulation at 0.1 nM 5α-DHT and reducedcell proliferation to normal levels at higher concentrations. Simard etal. (Simard, J., et al., Cancer Res., 51:4336-4341 (1991)) observedstimulation of cell proliferation at 0.5-1 nM 5α-DHT after a 10 dayexposure and an increase in apolipoprotein D secretion at 5 nM to 1 μM.Other investigators have studied the effects of the non-metabolizable,synthetic androgen R1881. Growth stimulation was observed from 0.1 nM to0.1 μM (Berns, E.M.J.J., et al., Prostate, 9:247-259 (1986); andLangeler, E. G., et al., Prostate, 23:213-223 (1993)). At 1 nM andhigher concentrations, stimulation of PAP secretion was observed(Langeler, E. G., et al., Prostate, 23:213-223 (1993)). Thesedifferences in hormonal response in different studies may be due todifferent culture conditions, different sublines or vastly differentpassages of LNCaP that may have been used in different studies. Otherreasons may be the rapid metabolism of 5α-DHT in cell cultures. Berns etal observed that ˜50% of the added (25 nM) 5α-DHT is metabolized within2 hours in prostate cell cultures. They also showed that theproliferative response of cells also depended on the cell number plated(Berns, E.M.J.J., et al., Prostate, 9:247-259 (1986)).

b. Assay for responsiveness to androgens

A microplate assay for establishing effects of androgens on the growthof cell lines was used. This assay has been routinely used by Webber andother study growth modulation (Cai, D., et al., Exp. Cell Res.,192:366-372 (1991)). 5,000 or 10,000 cells were plated/well of a 96-wellplate in 200 μl of serum-free K-SFM. After 24 hours, medium was changedto media containing a range of concentrations of 5α-DHT. Mediumcontaining 5α-DHT was changed every 48 hrs. Test plates were taken after7 and 9 days of treatment and stained with the protein-binding dye,methylene blue. Bound dye was released with 1% sodium dodecyl sulfate(SDS) and absorbance measured at 620 nm with a microplate readeraccording to a method described earlier (Cai, D., et al., Exp. CellRes., 192:366-372 (1991)). The results are shown in FIG. 5.

The prostatic epithelial origin of PWR-1E cells was confirmed byexamining PSA and PAP expression, responsiveness to androgens and thepresence of androgen receptor. It should, however, be noted that theimmortalized neonatal prostatic cell line developed by Kaighn et al(Kaighn, M. E., et al., Cancer Res., 49:3050-3056 (1989)), does notexpress PSA or PAP and their prostatic carcinoma cell line also does notexpress PSA, expresses very low levels of PAP and is unresponsive toandrogens (Kaighn, M. E., et al., Cancer Res., 49:3050-3056 (1989)).PWR-1E cells showed a moderate growth response to 5α-DHT treatment.Additional experiments are in progress using other, more stableandrogens.

c. Differentiation

Differentiated, secretory prostatic epithelial cells secrete PSA andisozyme II PAP (Lin, M.-F., et al., Cancer Res., 52:4600-4607 (1992)).These proteins can be used as specific markers for establishing that theimmortalized cells are: 1) of prostatic epithelial original, and 2) thatthey are capable of secreting these proteins, as they do in vivo. BothPSA and PAP are androgen responsive and their secretion can be modulatedin androgen-responsive cells in vivo and in vitro, e.g., by exposure to5α-DHT. The expression of these two proteins in response to "Mibolerone"a non-metabolizable androgen, and of androgen receptor in PWR-1E cellsis being examined to demonstrate beyond doubt their prostatic epithelialorigin. The primary and secondary cultures from which PWR-1E cells werederived, were positive for PSA.

It is important to note that a moderate growth response and PSA and PAPexpression in response to 5α-DHT of cultures grown in serum-free mediummay be due to the presence of epidermal growth factor (EGF) in themedium which is invariably a component of serum-free media. EGF is apotent inducer of cell proliferation in epithelial cells. It has beenshown that addition of EGF to medium containing 5% fetal calf serumcontributes to the loss of steroid binding (Schuurmans, A. L., et al.,Int. J. Cancer, 42:917-922 (1988)). Experiments on 5α-DHT growthresponse and PSA and PAP expression were conducted in K-SFM medium(GIBCO, Grand Island, N.Y.) which contains 5 ng/ml EGF. Theseexperiments need to be done using less or no EGF, if possible. Thiseffect of EGF can be explained by the observation that cellproliferation and expression of differentiated function in response toandrogens, such as 5α-DHT, have an inverse relationship.

PWR-1E cells express SV-40 large-T antigen. They were non-tumorigenicwhen tested for tumorigenicity in nude mice. They grow in a serum-freedefined medium with a doubling time of about 40 hours.

d. Plasminogen activator (PA) expression

Differentiated prostatic epithelial cells normally secret bothtissue-type (t-PA) and urokinase-type (u-PA) PAs which form an importantcomponent of the seminal fluid (Webber, M. M., et al., Retinoic acidmodulates urokinase-type plasminogen activator expression in DU-145human prostate carcinoma cells (In preparation) (1994)). These enzymescan serve as an additional marker for prostatic epithelial function,especially as urokinase expression responds to testosterone exposure andcould be used for early detection of prostate cancer. Expression ofplasminogen activators and type IV collagenases in PWR-1E cells issimilar to that in normal prostatic epithelium.

e. Assays for plasminogen activator activity

i. Zymography

Conditioned media from cultures of normal prostatic epithelium, PWR-1Ecells and DU-145 human prostate carcinoma cells were subjected toSDS-PAGE zymography. Results show that the level of u-PA activity inPWR-1E cells is similar to that in normal cells, whereas, the malignantcells express considerably higher levels of u-PA.

ii. Chromogenic substrate assay

The activity of urokinase (u-PA) in conditioned media was alsodetermined by a chromogenic substrate assay where the increase inabsorbance of the free chromophore generated was measured and comparedto the original u-PA specific substrate per unit time at 405 nm. Atexcess substrate concentration, the rate of absorbance increases due tothe amount of chromophore released and is linearly related to enzymeconcentration (Webber, M. M., et al., A. Retinoic acid modulatesextracellular urokinase-type plasminogen activator activity in DU-145human prostate carcinoma cells, In preparation, (1994)).

f. Type IV collagenase expression

Secretion of type IV collagenase was examined using SDS-PAGE zymographyusing gelatin or type IV collagen as substrates and samples ofconditioned medium from PWR-1E cells cultures were tested. Preliminaryresults show that collagenase expression in PWR-1E cells is similar tothat in normal prostate epithelial cells. These results provide one morenormal characteristic maintained by PWR-1E cells.

EXAMPLE 3

1. Isozyme analysis

Human origin of PWR-1E cells was established by isozyme analysis. PWR-1Ecells have the following isozyme profile: LDH, human (lactatedehydrogenase); NP, human (purine nucleoside phosphorylase); G6PD, B(glucose-6-phosphate dehydrogenase); PGM1, 1 (phosphoglucomutase-1);PGM3, 1 (phosphoglucomutase-3); ESD, 1 (esterase D); Me-2, 0 (malicenzyme, mitochondrial); AK-1, 1 (adenylate kinase); GLO-1, 2(glyoxalase-1).

2. Karyotype analysis

A complete chromosome analysis was performed to establish that the cellsare of human male origin. Chromosome number, presence of the Ychromosome, stable translocations, marker chromosomes and G-bandingpattern were examined.

The cell line is aneuploid human male, with most chromosome counts inthe hypotriploid range. A normal Y chromosome is present in about 20% ofmetaphases. Normal chromosomes #1, #2, #9, #14, #16, #17, #18, and #22are most often trisomic; chromosomes #5, #15, #20, and #21 are usuallytetrasomic; while the remainder of normal chromosomes are usuallydisomic. Cytogenetic alterations, other than copy number, are notprominent: most chromosomes are normal. Cells other than those of cellline PWR-1E p17 were not detected in the culture. They are hypotriploidand carry a Y chromosome. Twenty percent (20%) of the cells contain anormal Y chromosome.

3. Cytopathic effects assay in Vero cells

The PWR-1E cells were tested for free virus production, using this assayin the sensitive Vero cells (Rhim, J. S., et al., Science, 227:1250-1252(1985)). This assay was performed to establish that PWR-1E cells were anon-producer cell type. Only those immortalized cells that have anintegrated vital genome and are not shedding the virus are useful. TheVero cell line is derived from the kidney of a normal, adult Africangreen monkey. Vero cells (ATCC CCL 81) are permissive for the growth ofSV40 and adenoviruses which results in a lytic infection of cells,producing cytopathic effects (CPE).

Vero cells were grown in Dulbecco's MEM with 5% fetal bovine serum. Tenthousand (10,000) Vero cells were plated per well in 6-well plates. At50% confluency, cultures were inoculated, in duplicate, with 500 μl ofundiluted and diluted (1:2, 1:4, 1:8, 1:10), four-day conditioned mediumfrom PWR-1E cells. Cells were maintained for 21 days and observedperiodically for cytopathic effects. Conditioned medium from PWR-1Ecells did not cause CPE in Vero cells, hence, they are a non-producercell line.

4. Growth curves

Cells were plated in 96-well plates at different densities varying from625 cells to 40,000 cells per well in triplicate. Medium was changedevery 8 hours. Plates were taken at 48 hr. intervals, beginning with 2days after plating, fixed, stained and prepared for reading absorbanceas described earlier (Cai, D., et al., Exp. Cell Res., 192:366-372(1991)). Growth curves for PWR-1E cells are shown in FIG. 4. These cellshave a doubling time of about 40 hrs.

5. Immunoperoxidase and immunofluorescence methods

Expression of cytokeratins, desmin, Factor VIII, PSA and PAP proteinexpression was detected using monoclonal antibodies (MoAbs) andavidin-biotin immunoperoxidase (Vector ABC kit No. PK-6102) orimmunofluorescence methods (Antibodies: A Laboratory Manual., New York:Cold Spring Harbor laboratory, pp. 359-413, (1988)). MoAbs are availablefor all of the antigens to be tested, from Sigma, St. Louis, Missouri(CK 8, No. C-5301; CK 18, No. C-8541,) and Dako, Carpinteria, Calif.(Desmin, No. D-1033; Factor VIII, No. F-3520, PSA, No. M-750). Twodifferent sets of cultures were tested for each cell line. Appropriatecontrols employing non-immune serum were used. The results are shown inFIGS. 6A to 6D.

6. Agar assay for anchorage independence

Normal cells do not form colonies in soft agar. Immortalized PWR-1Ecells did not form colonies in agar. Thus, like normal cells, they areanchorage dependent, while the malignant cells will be expected to formcolonies. DU-145, human prostate carcinoma cells, form colonies in agar,and are used as a positive control. Colonies of anchorage independentcells appear in 3-5 weeks.

7. Growth in nude mice

Ten (10) million cells were injected subcutaneously in 3 mice. Mice wereexamined periodically for tumor development and were maintained for fourmonths. After four (4) months, none of the 3 mice injected with PWR-1Ecells had developed tumors at the site of injection. Hence, PWR-1E cellsare not tumorigenic.

8. Lack of invasive ability

It was anticipated that the immortalized cells would not be invasive inan in vitro invasion assay using our constituted basement membrane.Results show that PWR-1E cells are non-invasive as compared to carcinomacell lines.

a. In vitro assay for invasive potential of cells

This assay involves the use of Boyden chambers where cells plated on topof a porous filter coated with a reconstituted basement membrane"matrigel," in the upper chamber were allowed to invade and migratethrough matrigel for a period of 6 hrs. The lower chamber containedfibroblast conditioned medium as a chemo-attractant. The speed ofmigration correlates with the invasive potential (Webber, M. M., et al.,A. Retinoic acid modulates extracellular matrix degradation mediated byproteases secreted by prostatic carcinoma cells, in preparation, 1994;Kleinman, H. K., et al., Biochemistry 25:312-318 (1986); Albini, A., etal., Cancer Res., 47:3239-3245 (1987)). Cells migrate to the undersideof the filter, which are then stained on the filter with HEMA 3 (CurtinMatheson, Houston, Tex., USA). The nuclear stain is then extracted with0.1N HCl and absorbance is measured using a Microplate reader(Grotendorst, G. R., Methods Enzymol., 147: 144-152 (1987)). The numberof cells migrated shows a correlation with absorbance.

9. Expression of viral antigens

The expression of SV40 large-T antigen in PWR-1E cells was examined.SV40 immortalized cells have been shown to carry the immortalizing,large-T antigen gene (Gish, W. R., et al., J. Virol., 61:2864-2876(1987); and Reddel, R. R., et al., Cancer Res., 48:1904-1090 (1988)).PWR-1E cells express the large-T antigen.

10. Prostate pathology

An analysis of the pathology of the prostate shows that the incidence oflatent carcinoma of the prostate is about the same worldwide (Webber, M.M., et al., In Vitro Models for Cancer Research, Vol. V., pp. 3-24, BocaRaton, Fla.:CRC Press. (1988)). However, the high incidence Americanmale population shows a higher percentage of more aggressive,invasive-type latent carcinomas which develop into clinical carcinomas,compared to low incidence populations which show less aggressive,non-invasive-type, latent carcinomas. New evidence indicates that theprecancerous lesions appear at a young age in men in their twenties andthirties (Bostwick, D. G., J. Cell. Biochem., 164:10-19 (1992)). Thissuggests that there is a long latent period of 20 to 30 years beforecancer develops. Early changes during this period are an excellenttarget for cancer prevention. The best cure for cancer is prevention.The availability of immortalized cell lines makes it possible to testagents which could be used for cancer prevention, starting at a youngage when cancers appear to first originate.

EXAMPLE 4

1. Immortalization and the phenomenon of senescence

Senescence appears to be characteristic of all normal human cells invivo and in culture. SV40 transformed human cells typically haveextended in vitro lifespan but eventually reach a stage referred to as"crisis", where the cultures become static and cell proliferation isgreatly reduced or ceases. The parent cultures of PWR-1E cells did notexperience this crisis phase because cells infected with the hybridvirus escape the crisis stage.

Clone formation and immortalization were achieved at a high efficiencybecause the primary cells used for virus infection were grown in aserum-free medium. Many epithelial cells, especially epidermal-likecells are usually induced to terminally differentiate inserum-supplemented medium because TGF-βis a major serum-derivedinhibitor (Stoner, G. D., et al., Cancer Res., 51:353-371 (1991)). As aresult of this differentiation, many virus-infected cells wouldterminally differentiate and would be lost. Establishment of wellcharacterized, human, non-malignant cell lines allows detailedinvestigation of many aspects of the normal prostatic epithelial cellphysiology and of prostate tumors. Such immortalized and wellcharacterized human prostate cell lines are not presently available.PWR-1E provides a useful tool to study the biology and the pathology ofadult prostatic epithelial cells, specifically to understand the stepsleading to prostatic transformation.

Malignant transformation of mammalian cells requires at least twoseparate functions i) the establishment function is concerned withimmortalization of cells and ii) transformation function is required forfull expression of an oncogenic phenotype (Ruley, H. E., Nature, 304:602-606 (1983)). For example, Polyoma virus middle-T and the T24 Harveyras 1 gene are unable individually to transform primary baby rat kidneycells. However, adenovirus early region E1A gene provides functionsrequired by these genes to transform cells. Also, early region E1B isunable to transform cells in the absence of E1A. E1A immortalizes andE1B transforms, therefore, both are needed for malignant transformation.The immortalization step enables cells to respond to growth factors andto the action of transforming proteins (Ruley, H. E., Nature, 304:602-606 (1983)).

2. Some examples which demonstrate the cooperate nature of immortalizingand transforming genes

Human keratinocytes were immortalized by infection with AD12-SV40 hybridvirus. These cells are not tumorigenic. Subsequent infection withKirsten murine sarcoma virus, i.e., with Ki-ras oncogene resulted in theacquisition of neoplastic properties. These results support themultistep process of carcinogenesis (Rhim, J. S., et al., Science,227:1250-1252 (1985)). Neither control nor Ki-MSV infected cells couldbe propagated serially beyond 2 or 3 subcultures. In contrast, AD12-SV40infection led to the appearance of actively growing colonies by 3 to 4weeks. By week 6, T-antigen was revealed in the nuclei. Cells containedSV40 transcripts but not AD12. No transcripts were detected fromadenovirus E1A and E1B regions in these transformed human cells (Rhim,J. S., et al., Science, 227:1250-1252 (1985)). Infection of primary babyrat kidney cells with adenovirus 5 resulted in increase in cellproliferation. These cells produced a growth factor which stimulated thegrowth of primary cultures (Quinlan, M. P., et al., Proc Natl. Acad.Sci. USA, 84:3283-3287 (1987)). Results suggest that E1A exerts itstransformation potential through interactions with products of the tumorsuppressor genes (Whyte, P., et al., Cell, 56:67-75 (1989)).

Chimpanzee skin fibroblasts immortalized with AD12-SV40 virus containedAD12 and SV40 antigens and produced virus. However, at passage 9, oneline became non-producer and did not show virus-specific antigens.Hybrid virus could not be rescued from these cells by co-cultivationwith Veto cells ((Rhim, J. S., et al., Proc. Natl. Acad. Sci. USA78:313-317 (1981)).

In summary, we have established an AD12-SV40 immortalized cell linePWR-1E, derived from human prostatic epithelium. PWR-1E cells expressmany characteristics of normal epithelial cells and are non-tumorigeniccells. Cells show a growth response to 5α-DHT. Experiments to accomplishfurther characterization are in progress. These cells provide animportant model for studies on prostate carcinogenesis.

The following description demonstrates the significance of the novelnon-malignant cell lines

The applications for the cell lines are in the areas of causes,prevention and treatment of prostate cancer and of benign prostatichyperplasia (BPH); role of diet and nutrition in cancer; aging research;cell physiology; studies on the mechanisms of secretion; cell biology;biochemistry; studies on cell differentiation; developmental biology;and all biomedical sciences.

1. Acquisition of normal human tissue

Specimens of normal human prostate are very difficult to obtain becauseof the availability and consent concerns. Since PWR-1E cells retain manycharacteristics of normal cells, they may be used in experiments inplace of normal cells, which are generally not available to mostinvestigators.

2. Human cell models

Use of human cell models is considered much more appropriate than usingrat or mouse cells, data from which then have to be extrapolated to thehuman condition.

3. In vitro models

Use of cell models for research is preferred as much as possible inorder to avoid unnecessary use of whole animals.

The cell lines can be used for research in the following and otherareas:

4. Cancer detection and diagnosis

As models for the discovering new markers for early detection ofprostate cancer and BPH.

5. Cancer treatment

(a). The cells, transformed as described above, can be used as cellsystems for the discovery of new drugs such as cisplatin and taxol forthe treatment of prostate cancer and BPH.

(b). As cell models for examining the efficacy of single drugs andcombination of drugs and/or radiation for the treatment of prostatecancer.

(c). As cell systems for identification of anti-invasive agents, such asprotease inhibitors, i.e., inhibitors of urokinase and type IVcollagenase, and other agents which can specifically prevent invasion,including anti-motility factors, and thus can be used to prevent cancerfrom invading the neighboring tissue.

(d). As cell models for the identification of anti-metastasis agents,which can specifically prevent cancer from spreading to distant organs.

(e). As cell models for the identification of anti-angiogenic agents,which can specifically prevent growth of new blood vessels into thetumor.

6. Cancer prevention

As models for the discovery and testing of agents which may have theability to prevent prostate cancer and benign prostatic hyperplasia.

7. Basic Research

The cell models can be used for a large variety of studies, for example:

(a). The immortalized human prostatic cells are especially useful forstudying the etiology and multistep process of carcinogenesis in theprostate. This immortalized prostatic cell line, capable of growth inserum-free medium, is useful for studying neoplastic transformation andthe multistep process of carcinogenesis involving initiation andpromotion in the prostate and for investigating the action of putativeprostatic carcinogens. This may include oncogenes, chemical carcinogensand promoters, and radiation. These cells can also be used for theidentification of carcinogenic agents and the process of cancerdevelopment in the human prostate.

(b). For the identification of agents which cause benign prostatichyperplasia and the process of BPH development.

(c). For the identification of specific genetic defects, in themulti-step process of carcinogenesis, which lead to the development ofprostate cancer.

(d). For studies on why prostate cancer does not respond well tochemotherapy.

(e). For studies on the mechanisms of drug resistance in prostate cancerand how to circumvent resistance.

(f). Cells immortalized with this hybrid virus can be used to study:

i. The interactions between the tumor suppressor gene encoded host cellproteins, such as p53 and pRB and the viral immortalizing genes such asadenovirus E1A and SV40 large-T antigen. As shown in Table 1, viralproteins are able to bind and sequester the suppressor proteins, thus,allowing uncontrolled growth. The E1A and SV40 large-T antigens preventthe suppressor proteins from performing their normal function, which isto suppress cell proliferation. Loss or abnormal expression of p53 orpRB is associated with increased risk for cancer.

ii. The role played by suppressor genes in prostate cancer.

iii. The multistep process of carcinogenesis in the human prostate.

iv. Escape from senescence, i.e., immortalization. This is important notonly for improving our understanding of the aging process but also ofcancer since malignant transformation results in the development ofimmortal cells.

                  TABLE 1                                                         ______________________________________                                        Viral protein  Suppressor gene protein                                        ______________________________________                                        SV40 large T antigen                                                                         p53, pRB                                                       E1A of adenovirus                                                                            pRB                                                            E1B of adenovirus                                                                            p53                                                            ______________________________________                                    

Normal cells undergo senescence and are lost. However, immortalizationwith AD12-SV40 allows a study of escape from aging and death. SV40infected human cells go through a "crisis" phase which may last a yearor more, whereas, cells infected with the hybrid virus escape thiscrisis phase and clones of immortalized cells can be selected rapidly.Since prostate cancer incidence increases with age, this cell modelwould be especially useful for studies on aging and acquisition ofimmortality.

(g). Basic understanding of prostate physiology and its role inreproduction.

8. Mechanisms of tumor progression

Tumor progression itself is a multi-step process which includesinvasion, metastasis, angiogenesis and the development of drugresistance. The expression of proteases such as urokinase andcollagenase, is associated with progression. The cells can be used forstudies, for example:

(a). The mechanism of tumor progression and the associated genetic andepigenetic changes.

(b). Increased expression of the above mentioned proteases and theirappearance in the blood of cancer patients may serve as anindicator/marker of the advanced stage of the disease. Thus, the celllines can be used for the identification of agents which decrease ormodulate protease activity and thus, may be useful cancer prevention,intervention and therapeutic agents. These agents include anti-invasiveand anti-metastatic agents. Levels of these proteases in the blood couldalso be used as indicators of treatment efficacy.

(c). Tumor progression is also associated with angiogenesis. These celllines can be used for the identification of anti-angiogenic agents.

9. Models for risk assessment of reproductive toxicity

Reproductive toxicity may result in infertility in men, spontaneousabortions in their female partners and birth defects in their offspring.This cell line and derivatives thereof, can be used as:

(a). A model for identification of reproductive toxins which may besecreted into the seminal fluid and thus, damage sperm, resulting inabortions and birth defects.

(b). A model for detection of exposure to environmental and industrialreproductive toxins.

(c). A model for detection of reproductive toxicity in men exposed tochemicals in chemical warfare.

(d). A model for infertility and for birth defects in the offspring ofmen exposed to toxic chemicals in the industry and in chemical warfare.

(e). A model for studies on organ specific toxicity, and especially forstudies on the transport of toxins into the seminal fluid by prostaticcells. This cell model would be useful for studying uni-directionaltransport and secretion of toxins into the seminal fluid and theireffect on sperm. For instance, evidence is accumulating to suggest anassociation of pesticides with birth defects and cancer. Some of thesetoxins may be passed to infants via human milk. Others may damage spermby their presence in the prostatic fluid which provides a vehicle forsperm. The availability of well characterized human prostatic epithelialcell lines will provide useful models for studies on infertility andbirth defects.

10. Cells to be used as "normal" controls, when samples of humanprostate are not available

These cells retain many characteristics of normal epithelial cells,e.g., normal cytokeratin expression of cytokeratins 8 and 18, absence ofdesmin or Factor VIII expression, contact inhibition and monolayerformation in culture, response to 5-α dihydrotestosterone, anchoragedependence and inability to grow in agar, inability to form tumors innude mice and normal protease profiles. Since PWR-1E cells express manyof the characteristics of normal differentiated prostatic epithelialcells, they can be used for certain studies in place of normal humanprostatic epithelial cells, which are very difficult to acquire. This isa useful model for those studying the processes of cell secretion, forexample, in cystic fibrosis patients.

11. Studies on uni-directional transport of secretory proteins

PWR-1E cells are useful for studying such differentiated functions asuni-directional transport and secretion of prostate specific proteins.

12.Identification of potential chemotherapeutic drugs

These cells are useful for screening chemicals suitable for thetreatment of cancer and related diseases, by growing them in vitro inmedium containing the chemical to be tested and then, after a suitableperiod of exposure, determining whether and to what extent cytotoxicityhas occurred, e.g. by trypan blue exclusion assay or related assays(Patterson, Methods Enzymol. 58:141 (1979)), or by growth assays such ascolony forming efficiency (MacDonald et al., Exp. Cell. Res. 50:417(1968)), all of which are standard techniques well known in the art.

Similarly, potential chemotherapeutic drugs may be screened in vivo insuitable animal models. The tumorigenic or nontumorigenic human prostateepithelial cell lines are injected subcutaneously into athymic nudemice. Potential chemotherapeutic drugs are injected at various timeintervals and doses. Animals are examined weekly for the presence oftumors for a period of four or more months.

13. Studies of metabolism of carcinogens and other xenobiotics

Carcinogens and other xenobiotics may be added to the growth medium ofcultures of these cells and then the appearance of metabolic products ofthese compounds may be monitored by techniques such as thin layerchromatography or high performance liquid chromatography and the like.The interactions of the compounds and/or their metabolites with DNA canthen be examined.

14. Studies of DNA mutagenesis

Substances known or suspected to be mutagens may be added to the growthmedium of cultures of PWR-1E cells and then mutations may be assayed,e.g., by detection of the appearance of drug resistant mutant cellcolonies (Thompson, Methods Enzymol., 48:308 (1979)). Similarly,cell-mediated DNA mutagenesis in PWR-1E cells can be examined byco-cultivating them with a cell type known to have the ability toactivate a procarcinogen and, thus, release mutagenic metabolites intothe culture medium (Hsu et al., Proc. Natl. Acad. Sci. USA 75:2003(1978)).

15. Studies of chromosome damaging agents

Substances known or suspected to cause chromosomal damage may be addedto the culture medium of these cell lines, and then the extent ofchromosomal damage may be measured by techniques such as measurement ofthe frequency of sister chromatid exchange (Latt et al. In: Tice, R. R.and Hollaender, A., Sister Chromatid Exchanges, New York: Plenum Press,pp. 17-40, (1982)).

16. Studies of malignant transformation

By chemical, physical and vital agents, and transferred genes includingoncogenes and high molecular weight genomic DNA from tumors, usingstandard assays such as tumor formation in athymic nude mice.

17. Studies of cellular responses to growth factors and production ofgrowth factors

Identification and purification of novel growth factors important forgrowth and differentiation of human prostate epithelial cells. Thesecells are particularly useful for such an application since they grow inserum-free media. Therefore, responses to growth factors can be studiedin precisely defined growth media and any factors produced by the cellsmay be identified and purified without the complication of the presenceof serum.

18. Use of recombinant DNA expression vectors to produce proteins ofinterest: For example, the gene encoding a protein of therapeutic valuemay be recombined with controlling DNA segments (i.e. containing apromoter with or without an enhancer sequence), transferred into thecell (e.g. by polybrene transection) and then the protein produced maybe harvested from the culture supernatant or a cellular extract byroutine procedures well known in the art.

19. Characterization of cell surface antigens

The cells are incubated with an antibody against the cell surfaceantigen of interest, and then reacted with a second antibody which isconjugated to a fluorescent dye. The cells are then evaluated using afluorescence activated cell sorter to determine whether they arefluorescent and therefore possess the cell surface antigen.

20. Cell-cell hybrid studies for identification of tumor suppressoractivity (Stanbridge et al, Science 215:252-259 (1982))

To determine whether immortalized non-malignant cell lines contain tumorsuppressor genes, they are fused to malignant tumor cells. The presenceof tumor suppressor genes in hybrid cells is indicated by loss ofmalignant properties, i.e, loss of in vitro characteristics of cancercells, such as, density dependent inhibition of growth, growth in agar,stimulation of growth by autocrine secretion of growth factors andability to form tumors in athymic nude mice.

21. Identification of novel genes

Including transforming genes in naturally occurring cancers, growthfactor genes, oncogenes, tumor suppressor genes, using standardmolecular biological techniques (Davis et al., Methods in MolecularBiology, New York: Elsevier, pp. 167-189, (1986)) and techniques such ascDNA substraction cloning and the like.

22. Model for alternatives to animal testing

There is presently a great deal of concern about the use of animals fortesting products for human use. With the upsurge of interest in theAnimal Rights Movement, considerable emphasis is being placed on thedevelopment of cell culture models as alternatives to using animals fortesting. The drug and the cosmetic industry and manufacturers ofhousehold and industrial chemicals are interested in using cell culturemodels, especially those derived from human cells, to test for humantoxicity of their products.

A kit for screening carcinogenic or antineoplastic agents and for anyother usage as described herein supra, is easily assembled, comprisingthe cell line(s) of the present invention. Other components routinelyfound in such kits may also be included with instructions for performingthe test.

The cells of the present invention may be made malignant by exposure toa virus, transfected with an activated oncogene, radiation or tochemical carcinogens including, for example,N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea(NMU), tumor promoters such as and by promotion with12-O-tetradecanoylephorbol-13-acetate (TPA) or hormones. All of this iswell known to those skilled in the art.

The cell lines include cells of epithelial and fibroblast origin. Theepithelial cells include stem, basal, intermediate and differentiatedluminal cells. The prostatic cells can be from various donors and celltypes.

An application which is co-pending with the present application by someof the inventors herein describes HPV immortalized cells. This inventionis different than the present invention since a different virus wasused.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

We claim:
 1. A method for testing carcinogenicity of an agent comprisingculturing an immortalized adult human normal prostatic epithelial orfibroblast cell derived cell line free of other cell lines andcontaining DNA of adenovirus and simian virus 40 as a hybrid virus, thecell line having the identifying characteristics of the prostaticepithelial or fibroblast cell without the hybrid virus in addition tobeing immortalized by the hybrid virus with an agent suspected of beingcarcinogenic or exposing the sells to radiation, and determiningformation of an abnormal cellular mass by said cell line, the formationof the abnormal cellular mass being indicative of carcinofenicity ofsaid agent.
 2. The method of claim 1 wherein a normal tumor suppressorgene is introduced into the cell which results in a loss oftumorigenicity.
 3. A method of determining carcinogenicity promotingactivity of an agent comprising:a) culturing an immortalized adult humannormal prostatic epithelial or fibroblast cell derived cell line free ofother cell lines and containing DNA of adenovirus and simian virus 40 asa hybrid virus, the cell line having the identifying characteristics ofthe prostatic epithelial or fibroblast cell without the hybrid virus inaddition to being immortalized by the hybrid virus with an agentsuspected of being carcinogenic; and b) measuring a change in phenotypiccharacteristics of the cell line induced by the agent.
 4. The method ofclaim 3, wherein the phenotypic characteristic is increased urokinaseexpression, said increase being indicative if carcinogenicity of theagent.
 5. The method of claim 3, wherein the phenotypic characteristicis increased type IV collagenase expression, said increase beingindicative of promoting activity of the agent or the agent having theability to enhance tumor progression.
 6. The method of claim 1 whereinthe simian virus 40 is deposited as ATCC VR-239.
 7. The method of claim1 wherein the adenovirus if adenovirus 12 deposited as ATCC VR-863. 8.The method claim 1 wherein the adenovirus is adenovirus 12 and thesimian virus 40 is deposited as ATCC VR-863 and VR-239, respectively. 9.The method of claim 1 wherein the cell line is deposited as ATCC CRL11611.
 10. The method of claim 1 wherein the cell line has thephenotypic characteristics comprising: cytokeratins 8/18 positive,desmin negative, Factor VIII negative, contact inhibited, anchoragedependent, androgen responsive, plasminogen activator secretion normal,collagenase secretion normal, is non-tumorigenic, produces prostatespecific antigen and contains a hybrid virus of adenovirus and simianvirus
 40. 11. The method of claim 1 wherein the cell line producesprostate specific antigen.